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WO2018136435A1 - Compositions et procédés de traitement de maladies et de troubles du stockage lysosomial - Google Patents

Compositions et procédés de traitement de maladies et de troubles du stockage lysosomial Download PDF

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Publication number
WO2018136435A1
WO2018136435A1 PCT/US2018/013909 US2018013909W WO2018136435A1 WO 2018136435 A1 WO2018136435 A1 WO 2018136435A1 US 2018013909 W US2018013909 W US 2018013909W WO 2018136435 A1 WO2018136435 A1 WO 2018136435A1
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Prior art keywords
gld
metallothionein
mice
disease
incl
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PCT/US2018/013909
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Inventor
Alessandra Biffi
Eleonora CAVALCA
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Ospedale San Raffaele SRL
Boston Childrens Hospital
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Ospedale San Raffaele SRL
Boston Childrens Hospital
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Priority to CA3050691A priority Critical patent/CA3050691A1/fr
Priority to JP2019559007A priority patent/JP7225115B2/ja
Priority to CN201880018596.4A priority patent/CN110913872B/zh
Priority to US16/477,829 priority patent/US11548936B2/en
Priority to EP18741761.3A priority patent/EP3570670B1/fr
Priority to AU2018210853A priority patent/AU2018210853B2/en
Publication of WO2018136435A1 publication Critical patent/WO2018136435A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825—Metallothioneins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/825—Metallothioneins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders

Definitions

  • LSDs lysosomal storage disorders
  • CNS central nervous system
  • HCT hematopoietic stem cell
  • HSC hematopoietic stem cell
  • HSC GT hematopoietic Leukodystrophy
  • MLD Metachromatic Leukodystrophy
  • compositions and methods for the treatment or prevention of a lysosomal storage disorder e.g., Neuronal Ceroid Lipofuscinoses
  • a lysosomal storage disorder e.g., Neuronal Ceroid Lipofuscinoses
  • the methods involve replacing a patient' s endogenous microglia with either donor derived or engineered cells able to contribute to disease amelioration by different mechanisms, such as protein delivery or regulation of local inflammation and oxidative stress or others.
  • compositions and methods of treating a lysosomal storage disease or disorder in a subject involving increasing the level, expression, or activity of a metallothionein polyepeptide or polynucleotide in the subject relative to a reference.
  • the lysosomal storage disorder with CNS involvement is Neuronal Ceroid Lipofuscinoses, globoid leukodystrophy, GM1 gangliosidoses, Juvenile Hexosaminidase A Deficiency, Metachromatic Leukodystrophy, Mucopolysaccharidoses disorders, Multiple sulfatase deficiency, Tay-Sachs/GM2 gangliosidosis.
  • the subject is pre-selected by detecting an increase in the level of a metallothionein (MT) polynucleotide or polypeptide in a sample of the subject relative to a reference.
  • MT metallothionein
  • the metallothionein is one or more of metallothionein- 1 A (MTIA), metallothionein- IB (MTIB), metallothionein- IE (MTIE), metallothionein- IF (MT1F), metallothionein-lG (MT1G), metallothionein- 1H (MT1H), metallothionein- II pseudogene (MTlIp or MTE), metallothionein-lL (LT1L or MT1R), metallothionem-lM (MT1M or MT1K), metallothionein- IX (MT1X), metallothionein-2 (MT2), metallothionein-2A (MT2A), metallothionein-3 (MT3), and metallothionein-4 (MT4).
  • the method involves administering one or more MTs to the subject.
  • the method involves generating in a subject a sustained mixed hematopoietic chimerism in the brain and in the extra-CNS tissues with Hematopoietic Stem Cells (HSCs) encoding one or more MTs.
  • the method involves treating a subject having or being at increased risk of developing a lysosomal storage disorder, including by administering a Hematopoietic Stem Cell (HSC) that is one or more of CD34 + , CD38 " , where the HSC is administered intravenously (IV) or by Intra-cerebral Ventricular (ICV) Injection in combination with ablative conditioning.
  • HSC Hematopoietic Stem Cell
  • IV Intra-cerebral Ventricular
  • the isolated HSC is transformed with a vector expressing one or more therapeutic polypeptide or polynucleotide, where the HSC is one or more of CD34 , CD38 " .
  • the HSC are engineered with integrating vectors, i.e. lentiviral vectors, to express a one or more
  • the method involves ablating endogenous myeloid cells and microglia and/or their progenitors by a conditioning regimen and reconstituting the microglia by HSC engraftment in a subject.
  • the HSC is administered in combination with ablative conditioning.
  • the ablative conditioning comprises administering a cytotoxic agent to the subject.
  • the alkylating agent is busulfan.
  • the ablative conditioning is performed prior to administering the HSC.
  • agent any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • antibody refers to an immunoglobulin molecule which specifically binds with an antigen.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • alteration or “change” is meant an increase or decrease.
  • An alteration may be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%.
  • biological sample any tissue, cell, fluid, or other material derived from an organism.
  • capture reagent is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.
  • the terms “determining”, “assessing”, “assaying”, “measuring” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase “determining a level" of an analyte or “detecting” an analyte is used.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • detectable label is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • an effective amount is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • fragment is meant a portion of a protein or nucleic acid that is substantially identical to a reference protein or nucleic acid. In some embodiments the portion retains at least 50%, 75%), or 80%), or more preferably 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the term "purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%>, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • lysosomal storage disorder refers to any of a group of diseases resulting from abnormal metabolism leading to accumulation of a substrate (for example sulfatides, heparan sulphate, glycolipids, ceramide) in the lysosome.
  • a substrate for example sulfatides, heparan sulphate, glycolipids, ceramide
  • LSDs lysosomal storage disorders
  • LSDs lysosomal storage disorders
  • mucopolysaccharides are caused by lysosomal dysfunction usually as a consequence of deficiency of an enzyme required for the metabolism of lipids, glycoproteins (sugar-containing proteins) or so-called mucopolysaccharides.
  • marker any clinical indicator, protein, metabolite, or polynucleotide having an alteration associated with a disease, disorder, or condition.
  • microglia an immune cell of the central nervous system.
  • neurodegenerative disease refers to a disease characterized by the progressive loss of structure and/or function of neurons, including death of neurons.
  • increasing proliferation is meant increasing cell division of a cell in vivo or in vitro.
  • the terms "prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • subject refers to an animal which is the object of treatment, observation, or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.
  • reduces is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
  • reference is meant a standard of comparison or control condition.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e "3 and e "100 indicating a closely related sequence.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 42° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al.,
  • telomere a compound that recognizes and binds a molecule (e.g., polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.
  • a compound e.g., peptide
  • molecule e.g., polypeptide
  • the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • FIG. 1 A, FIG. IB, FIG. 1C and FIG. ID show disease model selection for preclinical testing of MTs as therapeutic agents in LSDs.
  • FIG. 1A shows representative pictures of MT immunoreactivity in post-mortem brain samples from GLD, MLD, NPC and NCL patients, as indicated. Grey and white matter (GM and WM) are shown for leukodystrophies, while only GM is shown for NPC and NCL. MT immunoreactivity is primarily associated to astrocytes in both cortex and white matter. Neurons showed MT immunoreactivity in NCL brain samples only (*), while MT positive histiocytes (#) were only observed in MLD. 40X magnification.
  • FIG. IB is a graph that provides relative abundance of Lrp2 mRNA in brain of LSDs with neurologic involvement, compared to age-matched normal donor (ND) samples.
  • One-way Anova with Bonferroni post-test, ** P value ⁇ 0.01.
  • FIG. ID is a graph showing MT1 mRNA expression levels in LSD mouse models.
  • FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, FIG. 2F, FIG. 2G, and FIG. 2H show phenotypic effects of MTs in the GLD and INCL animal models.
  • FIG. 2A is a graph showing MT brain expression in naive and MT-transgenic Gale-/- and
  • FIG. 2B shows representative confocal images of the pons region of a MT-GLD mouse at P D36 stained for astrocytes (GFAP-red), Metallothioneins (MT -green) and DAPI, confirming MT specificity of expression in astrocytes. 20X (left) and 40X (right) magnification.
  • FIG. 2C shows representative confocal images of the pons region of a GLD and of a MT- GLD mouse at P D36 stained for microglia (IBA-red), Metallothioneins (MT- green) and DAPI, showing few MT-positive cells co-localizing with microglia signal in both animals and more intense MT staining in the MT-GLD sample. 20X magnification.
  • FIG. 2D and FIG. 2E depict experiments in which an MT1 transgenic (over-expressing) mouse was crossed with either Gale-/- or Pptl-/- mice, which are animal models of the lysosomal storage diseases globoid cell leukodystrophy (or Krabbe disease) and neuronal ceroid
  • FIG. 2D provides a Kaplan-Meier survival curve of MT-GLD and GLD mice. Data were analyzed by Log-Rank (Mantel-Cox) test; P value ⁇ 0.0001.
  • FIG. 2E provides a Kaplan-Meier survival curve of MT-INCL and INCL mice. Data are analyzed by Log-Rank (Mantel-Cox) test; P value ⁇ 0.0001. Survival curves of naive and MT -transgenic Gale-/- (FIG. 2D) and Pptl-/- (FIG. 2E) mice were generated showing a survival advantage of the affected transgenic animals (over-expressing MTs) over the not transgenic affected mice.
  • Bonferroni correction * P value ⁇ 0.05, *** P value ⁇ 0.001.
  • FIG. 2G is a graph depicting correlation of survival data of FIG. 2D and MT levels (expressed as fold to WT levels) presented in FIG. 2A, including both GLD and MT-GLD data sets.
  • the figure represents the maximum survival of the natural occurring disease model interpolated with MT expression levels in the same animals, identifying the minimum MT level associated to survival gain.
  • the range of MT levels detected in GLD and MT-GLD mice is here also shown.
  • FIG. 2H is a graph depicting correlation of survival data of FIG. 2E and MT levels (expressed as fold to WT levels) presented in FIG. 2A, including both INCL and MT-INCL data sets.
  • the figure represents the maximum survival of the natural occurring disease model interpolated with MT expression levels in the same animals, identifying the minimum MT level associated to survival gain.
  • the range of MT levels detected in INCL and MT-INCL mice is here also shown.
  • FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, and FIG. 3G show modulation of anti-inflammatory, anti-apoptotic and anti- oxidative stress genes in MT-GLD and MT-INCL mice.
  • FIG. 3 A provides a hierarchical clustering representing differentially expressed genes, up- regulated and down-regulated, in the four tested groups. As shown in the bar, over- expression was visualized in shades of red and under- expression in shades of blue.
  • Transcriptome array was performed on cerebellar extracts from the following mice all analyzed at PND36: 3 WT, 3 MTtg, 3 MT-GLD and 3 GLD.
  • FIG. 3B, FIG. 3C, and FIG. 3D are graphs showing Ifi44 (FIG. 3B), Hpgd (FIG. 3C) and Casp4 (FIG. 3D) expression variations in LSD and MT-LSD brain samples.
  • FIG. 3C - FIG- 3D show fold expression changes in the indicated pairs, calculated from transcriptome analysis data; fold change of Ifi44 for MT-GLD vs GLD is -2,5528, P value ⁇ 0.001; fold change of Hpgd for MT-GLD vs GLD is -2,24130, P value ⁇ 0.001; fold change of Casp4 for MT- GLD vs GLD is -1,75215, P value ⁇ 0.01.
  • FIG. 3E shows representative pictures of nitrotyrosine (Nitro) staining in brain sections of WT, GLD and MT-GLD mice. Other regions were analyzed with the same expression pattern. All the animals were analyzed at PND36. Magnification 40X.
  • FIG. 3G (left panel) shows results presented as %DCFDA positive cells within total live myeloid cells. Mean with SEM.
  • G right panels Representative histograms, inclusive of the positive control (Co+, WT cells supplemented with H202). The expression of DCDFA is reduced in the trangenic affected mice as compared to naive affected controls.
  • ROS reactive oxygen species
  • FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G, and FIG. 4H show Purkinje cell loss is rescued in both MT-GLD and MT-INCL models.
  • FIG. 4 A, FIG. 4B, FIG. 4C, FIG. 4D, and FIG. 4E are representative images and graphs showing neuroprotective effect of MT in Gale-/- or Pptl-/- mice. Cerebellum sections from wild type, and naive and MT -transgenic Gale-/- (FIG. 4A, FIG. 4B) and Pptl-/- (FIG. 4C) mice are shown. Purkinje cells are detectable by positive Calbindin (CALB) staining (A) and by their morphological features at crystal violet (C). Purkinje cells were quantified and were shown to be markedly reduced in the diseased animals, but not in the disease MT transgenic mice.
  • CALB positive Calbindin
  • C crystal violet
  • FIG. 4A shows representative pictures of Calbindin staining on slices of the cerebellum of WT, GLD and MT-GLD mice. All the animals were analyzed at P D36.
  • FIG. 4C shows representative pictures of Parvalbumin staining in the cerebellum of WT, GLD and MT-GLD at P D36. Nuclei were stained with Topro III.
  • FIG. 4E shows representative pictures of Nissl staining to detect Purkinje cells, in the cerebellum of WT, INCL and MT-INCL. All the animals were analyzed at intermediate disease stage of 200 days.
  • FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H, FIG. 51, FIG. 5 J, and FIG. 5K show MTs induce an anti-inflammatory M2-like microglia phenotype in GLD and INCL.
  • FIG. 5 A shows representative pictures of GFAP staining in the pons region of WT, GLD and MT-GLD. All the animals were analyzed at PND36. 20X magnification.
  • INCL INCL
  • MT-INCL MT-INCL
  • FIG. 5C shows representative pictures of IB A staining in the pons region of WT, GLD and MT-GLD. All the animals were analyzed at P D36. 20X magnification.
  • INCL INCL
  • MT-INCL MT-INCL
  • FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H, and FIG. 51 are graphs showing the effect of transgenic MT over-expression on microglia phenotype in Gale-/- or Pptl-/- mice.
  • Microglia cells were sorted from the brain of wild type, naive affected and MT transgenic affected animals and tested for the expression of the listed genes.
  • Affected naive animals have a prevalent pro- inflammatory microglia phenotype (ILip and TNFa increased expression) that is reduced in MT transgenic affected animals.
  • CD206, ARG1, YM1 neuroprotective microglia phenotype
  • FIG. 5K shows representative pictures of the pons region of MTtg, GLD and MT-GLD mice showing co-localization of IB A signal with CD206. 40X magnification.
  • FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F, and FIG. 6G show MT delivery by AAV-PHP.B vectors ameliorates the GLD phenotype.
  • FIG. 6A is a graph that shows relative mRNA abundance of MT1 in HEK293T transduced with AAV- PHP.B (AAV) encoding 1 (AAV-MT) and 4 (AAV-4MT) MT-1 copies in two independent experiments (duplicate) and reported as fold to UT samples
  • AAV AAV encoding 1
  • AAV-4MT AAV-4MT
  • FIG. 6D is a graph depicting correlation of survival data of FIG. 6B and MT levels measured in the same mice, including both GLD and AAV-GLD data sets.
  • the figure represents the maximum survival of the natural occurring disease model interpolated with MT expression levels in the same animals, identifying the minimum MT level associated to survival gain.
  • the range of MT levels detected in GLD and AAV-GLD mice is here also shown.
  • FIG. 6E, FIG. 6F, and FIG. 6G include graphs that show relative mRNA abundance of Ifi44 (FIG. 6E), Hpgd (FIG. 6F), and Casp4 (FIG. 6G) in the brain of GLD mice injected IV with AAV-4MT or PBS, reported as fold to WT samples. * P value ⁇ 0.05 with unpaired t-test. Mean with SEM.
  • the invention features compositions and methods that are useful for the treatment and prevention of lysosomal diseases and disorders (e.g., Neuronal Ceroid Lipofuscinoses).
  • the methods involve increasing the level, expression, or activity of a metallothionein polypeptide or polynucleotide in the subject.
  • the methods involve ablating and/or reconstituting microglia.
  • the present invention is based at least in part on several discoveries described herein. It has been found that increasing levels of metallothionein polypeptides has a therapeutic benefit in subjects having lysosomal disease or disorder.
  • Lysosomal Storage Disorders are a broad class of inherited metabolic diseases caused by the defective activity of specific lysosomal enzymes.
  • Central nervous system (CNS) manifestations are present in roughly 50% of LSD patients and represent an unmet medical need for patients.
  • compositions and methods that explore the therapeutic potential of Metallothioneins (MTs), a newly identified family of proteins with reported neuroprotective roles, in murine models of two LSDs with CNS involvement.
  • MTs Metallothioneins
  • MTs Metallothioneins
  • MTs transgenic mouse over-expressing MT-1 in all tissues (strain B6.Cg- Tg(Mtl)174Bri/J, The Jackson Laboratory) were cross bred with the naturally occurring mouse model of Globoid Cell Leukodystrophy (GLD, also called Twitcher mouse) (Suzuki, 1995), being GLD a typical neuronopathic lysosomal storage disease caused by deficient activity of ⁇ - galactocerebrosidase (GALC), characterized by rapid and progressive demyelination and neuronal degeneration.
  • GLD Globoid Cell Leukodystrophy
  • Lysosomal Storage Disorders comprise more than 40 different diseases characterized by disruption of lysosomal function. Most of these conditions are characterized by unrelenting neurodegeneration. (Piatt FM, Nature 2014; 510(7503)) Lysosomal dysfunction leads to accumulation of incompletely degraded substrates causing mechanical damage of the cells and/or changes in cellular homeostasis that result in apoptosis. (Futerman AH et al., Nat Rev Mol Cell Biol 2004; 5(7)) In addition, perturbation of complex cell signalling mechanisms give rise to secondary structural and biochemical changes such as inflammation that contribute to tissue damage in LSDs. Central nervous system (CNS) manifestations are present in roughly 50% of LSDs and represent an unmet medical need for patients.
  • CNS Central nervous system
  • NCT02725580 ex vivo gene therapy, i.e. based on hematopoietic stem cells (i.e.
  • Lysosomal storage diseases include, without limitation, Neuronal Ceroid Lipofuscinoses (NCL), GM1 and GM2 Gangliosidosis, Alpha-mannosidosis, Globoid Cell Leukodystrophy (GLD), Neuronal Ceroid Lipofuscinosis (NCL), Metachromatic Leukodystrophy (MLD), Mucopolysaccharidoses disorders (MPSs), Multiple sulfatase deficiency (MSD), and Niemann- Pick Disease. Approximately 50% of LSDs have involvement of the CNS, as in the case of the examples listed above. A non-limiting list of exemplary SDs and their associated defective protein is provided at Table 1. Table 1. Lysosomal Storage Disorders (LSDs) and their associated defective protein
  • NCLs Neuronal Ceroid Lipofuscinoses
  • Neuronal Ceroid Lipofuscinoses are a class of inherited storage disorder that result in progressive neurological degeneration. Some variants, such as the late infantile NCL (LINCL), are caused by deficiency of a lysosomal enzyme. LINCL is caused by mutations in the CLN2 gene that result in the deficiency of TPP-I, a lysosomal enzyme that is responsible for degrading membrane proteins. Neurons are particularly sensitive to the lysosomal accumulation of this storage material, and individuals with LINCL have extensive, progressive neurodegeneration in all parts of the brain, resulting in a vegetative state and death by the age of 8-12 years.
  • LINCL late infantile NCL
  • MLD Metachromatic Leukodystrophy
  • Arylsulfatase A (ARSA) gene is a prototypical example of LSD with progressive accumulation of un-degraded substrates in the nervous system and secondary neuroinflammation and degeneration.
  • the genetic transmission of MLD is autosomal recessive and its overall incidence is estimated to be 1 :40,000-l : 100,000.
  • Globoid Cell Leukodystrophy GLD
  • Globoid Cell Leukodystrophy GLD
  • GLC Galactocerebrosidase
  • GalCer Galactosylceramide
  • the devastating neurodegenerative disorder is due to alterations in glycosphingolipid catabolism caused by GALC deficiency: the resulting accumulation of incompletely metabolized GalCer leads to progressive white matter disease which affects both the CNS and the Peripheral Nervous System (PNS).
  • PNS Peripheral Nervous System
  • GalCer GalCer
  • GalCer Peripheral Nervous System
  • GalCer GalCer
  • GalCer Peripheral Nervous System
  • GLD children can be treated when pre-symptomatic and below the age of 4- month-old by HCT from healthy compatible donors that delays disease onset and attenuates manifestations 20 .
  • HSC gene therapy was also proven to be potentially effective in GLD preclinical models 21 .
  • MPS Mucopolysaccharidoses
  • MPS I is divided into three subtypes based on severity of symptoms. All three types result from an absence of, or insufficient levels of, the enzyme alpha-L-iduronidase.
  • MPS I H also called Hurler syndrome or a-L-iduronidase deficiency
  • MPS I S Scheie syndrome
  • MPS I H-S Hurler-Scheie syndrome
  • MPS II Hunter syndrome or iduronate sulfatase deficiency
  • MPS III Sanfilippo syndrome, is marked by severe neurological symptoms.
  • Sanfilippo syndrome There are four distinct types of Sanfilippo syndrome, each caused by alteration of a different enzyme needed to completely break down the heparan sulfate sugar chain.
  • Sanfilippo A is the most severe of the MPS III disorders and is caused by the missing or altered enzyme heparan N-sulfatase. Children with Sanfilippo A have the shortest survival rate among those with the MPS III disorders.
  • Sanfilippo B is caused by the missing or deficient enzyme alpha-N acetylglucosaminidase.
  • Sanfilippo C results from the missing or altered enzyme acetyl-CoAlpha-glucosaminide acetyltransferase.
  • Sanfilippo D is caused by the missing or deficient enzyme N-acetylglucosamine 6-sulfatase.
  • MPS IV Morquio syndrome
  • MPS VI Maroteaux-Lamy syndrome
  • MPS VII Sly syndrome
  • Sly syndrome one of the least common forms of the mucopolysaccharidoses, is caused by deficiency of the enzyme beta-glucuronidase.
  • Neurodegenerative diseases are characterized by the progressive loss of the structure and/or function of neurons and/or neuronal cell death. Inflammation has been implicated for a role in several neurodegenerative diseases. Progressive loss of motor and sensory neurons and the ability of the mind to refer sensory information to an external object is affected in different kinds of neurodegenerative diseases.
  • a health care professional may diagnose a subject as having a neurodegenerative disease by the assessment of one or more symptoms of a
  • Non-limiting symptoms of a neurodegenerative disease in a subject include difficulty lifting the front part of the foot and toes; weakness in arms, legs, feet, or ankles; hand weakness or clumsiness; slurring of speech; difficulty swallowing; muscle cramps; twitching in arms, shoulders, and tongue; difficulty chewing; difficulty breathing;
  • muscle paralysis partial or complete loss of vision; double vision; tingling or pain in parts of body; electric shock sensations that occur with head movements; tremor; unsteady gait; fatigue; dizziness; loss of memory; disorientation; misinterpretation of spatial relationships; difficulty reading or writing; difficulty concentrating and thinking; difficulty making judgments and decisions; difficulty planning and performing familiar tasks; depression; anxiety; social withdrawal; mood swings; irritability; aggressiveness; changes in sleeping habits; wandering; dementia; loss of automatic movements; impaired posture and balance; rigid muscles;
  • a health care professional may also base a diagnosis, in part, on the subject's family history of a neurodegenerative disease.
  • a health care professional may diagnose a subject as having a neurodegenerative disease upon presentation of a subject to a health care facility (e.g., a clinic or a hospital). In some instances, a health care professional may diagnose a subject as having a neurodegenerative disease while the subject is admitted in an assisted care facility.
  • a physician diagnoses a neurodegenerative disease in a subject after the presentation of one or more symptoms.
  • MTs Metallothioneins
  • MTs are a family of metal-binding, non-enzymatic proteins known to exert an anti-oxidant and neuroprotective function in the diseased brain in several different pathological conditions.
  • MTs are released from astrocytes and re-uptaken by astrocytes themselves and neurons through the receptor Lrp2/megalin.
  • MT-transgenic disease models (of Neuronal Ceroid Lipofuscinosis - NCL, also known as Batten disease, and Globoid Cell Leukodystrophy - GLD, also known as Krabbe disease) were generated and analysed, characterized by the presence of constitutively high levels of MTs in all body tissues, including the CNS.
  • MTs exerted a beneficial effect on diseased mice phenotype.
  • This beneficial effect was also achieved when MT transcripts were delivered to mutant LSD mice by systemic administration of a MT-encoding AAV-PHP.B vector (Deverman BE. et al., Nat Biotechnol 2016; 34(2)), and was extensively related to anti-inflammatory, anti -oxidative and anti-apoptotic effects exerted by the MTs in the LSD CNS.
  • the compostions and methods disclosed herein indicate that exogenously delivered MTs could exert a therapeutic role in LSDs severely affecting the CNS by modulating disease-related mechanisms of neural damage.
  • the present invention provides methods of treating a lysosomal storage disease or disorder in a subject involving increasing the level, expression, or activity of a metallothionein polyepeptide or polynucleotide in the subject.
  • Metallothioneins are a family of small ( ⁇ 6- 7 kDa), heat-resistant proteins containing 25-30% cysteine residues that are evolutionarily highly conserved in a broad range of species from yeast to mammals. MTs are up-regulated by glucocorticoids, oxidative stress and a variety of heavy metals, such as copper, cadmium, mercury and zinc (Andrews (2000) Biochem. Pharmacol. 59, 95-104).
  • Isoforms range from MT- 1 to MT-4 and have slightly different amino acid composition.
  • MTs bind metals and protect against their toxicity, as was first demonstrated in aquatic species, such as fish, arthropods and molluscs from contaminated waters. Apart from binding heavy metals, MTs are considered to act as antioxidants, although by undetermined mechanisms. Thus MTs have been found to protect against apoptosis/necrosis induced by oxidative stress, etoposide, cisplatin, doxorubicin and X-irradiation (Cai et al. (2004) Toxicol. Lett. 146, 217-226; Chimienti et al. (2001) Free Radicals Biol. Med. 31, 1179-1 190; Wang et al. (2001) J. Pharmacol. Exp. Ther. 298, 461-468).
  • the MT transcript and protein described herein may be selected from, for example, metallothionein- 1 A (MT1A), metallothionein-lB (MT1B), metallothionein-lE (MT1E), metallothionein- IF (MT1F), metallothionein-lG (MT1G), metallothionein- 1H (MT1H), metallothionein- II pseudogene (MTlIp or MTE), metallothionein-lL (LT1L or MT1R), metallothionem-lM (MTIM or MT1K), metallothionein-lX (MTIX), metallothionein-2 (MT2), metallothionein-2A (MT2A), metallothionein-3 (MT3) or metallothionein-4 (MT4).
  • MT1A metallothionein- 1 A
  • MT1B metallothionein-lB
  • MT1E
  • NCBI protein accession numbers of the main members of the family are: P 005937 (MT1A); P_005938 (MT1B); P_783316 (MT1E); P_005940 (MT1F); P_005941
  • MT1G (MT1G); P_005942 (MTIH); P_789846 (MTIM); P_005943 (MTIX); P_005944 (MT2); P_005945 (MT3); and P_116324 (MT4).
  • Further NCBI accession numbers for MT1 A, MT1E, MT2A and MTE-MT1IP include: NM_005946, NM_075617, NM_005953 and
  • the present invention also provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising HSCs described herein to a subject (e.g., a mammal such as a human).
  • a subject e.g., a mammal such as a human.
  • one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof.
  • the method includes the step of administering to the mammal a therapeutic amount of a cell herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a cell described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • Engraftment of transplanted cells provides the expression or activity of a polypeptide or other therapeutic agent.
  • a deficiency in or loss of function of a lysosomal enzyme results in a lysosomal storage disorder.
  • Transplanted hematopoietic cells that express the therapeutic protein (e.g., an enzyme) either endogenously or via recombinant methods engraft and differentiate into microglia, thereby remedying the deficiency in the enzyme.
  • transplanted cells may serve as a vehicle for therapeutic polypeptides (e.g., one or more metallothionein polypeptides).
  • engraftment is enhanced by ablating existing microglia nd/or their progenitors (e.g., with alkylating agents).
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof.
  • Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).
  • a diagnostic test or opinion of a subject or health care provider e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like.
  • antibodies that specifically bind a marker are useful in the methods of the invention, including therapeutic methods.
  • the invention provides methods of ablating microglia involving contacting microglia with a nanoparticle having a capture molecule that specifically binds a marker of a microglial cell and containing a cytotoxic agent (e.g., an alkylating agent).
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Tetramers may be naturally occurring or reconstructed from single chain antibodies or antibody fragments. As used herein, the term "antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv fragments, linear antibodies, scFv antibodies, single- domain antibodies, such as camelid antibodies (Riechmann, 1999, Journal of Immunological Methods 231 :25-38), composed of either a VL or a VH domain which exhibit sufficient affinity for the target, and multispecific antibodies formed from antibody fragments.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab') 2 , as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85 :5879-5883; Bird et al., 1988, Science 242:423-426).
  • the antibodies of the invention comprise, without limitation, whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.
  • Unconventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062, 1995), single domain antibodies, single chain antibodies, and antibodies having multiple valencies ⁇ e.g., diabodies, tribodies, tetrabodies, and pentabodies).
  • Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies have the affinity and specificity of conventional antibodies although they are only half of the size of a single chain Fv fragment. The consequence of this unique structure, combined with their extreme stability and a high degree of homology with human antibody frameworks, is that nanobodies can bind therapeutic targets not accessible to conventional antibodies.
  • Recombinant antibody fragments with multiple valencies provide high binding avidity and unique targeting specificity to cancer cells.
  • These multimeric scFvs ⁇ e.g., diabodies, tetrabodies
  • offer an improvement over the parent antibody since small molecules of ⁇ 60-100kDa in size provide faster blood clearance and rapid tissue uptake See Power et al., (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting, 4, 47-58, 1999).
  • CEA Anti-carcinoembryonic antigen
  • Bispecific antibodies produced using leucine zippers are described by Kostelny et al. (J.
  • Single chain Fv polypeptide antibodies include a covalently linked VH: :VL heterodimer which can be expressed from a nucleic acid including VH- and VL-encoding sequences either joined directly or joined by a peptide-encoding linker as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and
  • an antibody is monoclonal.
  • the antibody is a polyclonal antibody.
  • the preparation and use of polyclonal antibodies are also known the skilled artisan.
  • the invention also encompasses hybrid antibodies, in which one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids may also be formed using humanized heavy and light chains. Such antibodies are often referred to as "chimeric" antibodies.
  • intact antibodies are said to contain "Fc” and "Fab” regions.
  • the Fc regions are involved in complement activation and are not involved in antigen binding.
  • An antibody from which the Fc' region has been enzymatically cleaved, or which has been produced without the Fc' region, designated an "F(ab' )2" fragment retains both of the antigen binding sites of the intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an "Fab"' fragment, retains one of the antigen binding sites of the intact antibody.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted "Fd.”
  • the Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.
  • Antibodies can be made by any of the methods known in the art utilizing a soluble polypeptide, or immunogenic fragment thereof, as an immunogen.
  • One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production.
  • the immunogen will facilitate presentation of the immunogen on the cell surface.
  • Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding polypeptides or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the polypeptide thereby generating an immunogenic response in the host.
  • nucleic acid sequences encoding human polypeptides or immunogenic fragments thereof can be expressed in cells in vitro, followed by isolation of the polypeptide and administration of the polypeptide to a suitable host in which antibodies are raised.
  • antibodies may, if desired, be derived from an antibody phage display library.
  • a bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins.
  • Phage display is the process by which the phage is made to 'display' the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.
  • Antibodies made by any method known in the art can then be purified from the host.
  • Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.
  • salt precipitation for example, with ammonium sulfate
  • ion exchange chromatography for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength
  • gel filtration chromatography including gel filtration HPLC
  • affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.
  • Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art.
  • the hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source.
  • Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or
  • the method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse.
  • the mammal may be primed for ascites production by prior administration of a suitable composition ⁇ e.g., Pristane).
  • Monoclonal antibodies (Mabs) produced by methods of the invention can be "humanized” by methods known in the art. "Humanized” antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins.
  • HCT Hematopoietic Cell Transplantation
  • hematopoietic stem and progenitor cells hematopoietic stem and progenitor cells
  • progeny can serve as vehicles for therapeutic molecule delivery across the blood brain barrier by contributing to the turnover of myeloid cell populations in the brain.
  • HSPCs hematopoietic stem and progenitor cells
  • the differentiation and functional characteristics of the cells reconstituted after transplantation are still to be determined, and in particular whether bona fide microglia could be reconstituted by the donor cell progeny post-transplant to be assessed.
  • HCT Hematopoietic Cell Transplantation
  • HSC Hematopoietic Stem Cell
  • BBB blood brain barrier
  • HSPCs and/or their progeny could contribute to the turnover of myeloid cell populations in the brain, possibly including microglia, whose crucial role in the progression and outcomes of these disorders has been extensively described.
  • cells derived from the transplant were proven to favorably affect the local environment, i.e. by releasing therapeutic molecules in the brain of transplanted mice or patients. This concept was demonstrated in patients affected by the demyelinating LSD metachromatic leukodystrophy treated by HSC gene therapy.
  • Metallothioneins are emerging for having a great potential as therapeutic agents for neurologic conditions.
  • DNA molecules obtained by any of the methods described herein or those that are known in the art can be inserted into appropriate expression vectors by techniques well known in the art.
  • a double stranded DNA can be cloned into a suitable vector by restriction enzyme linking involving the use of synthetic DNA linkers or by blunt-ended ligation.
  • DNA ligases are usually used to ligate the DNA molecules and undesirable joining can be avoided by treatment with alkaline phosphatase.
  • the invention includes vectors (e.g., recombinant plasmids) that include nucleic acid molecules (e.g., genes or recombinant nucleic acid molecules encoding genes) as described herein.
  • recombinant vector includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the recombinant vector was derived.
  • a recombinant vector may include a nucleotide sequence encoding a polypeptide, or fragment thereof, operatively linked to regulatory sequences, e.g., promoter sequences, terminator sequences, and the like, as defined herein.
  • regulatory sequences e.g., promoter sequences, terminator sequences, and the like.
  • one or more DNA molecules having a nucleotide sequence encoding one or more polypeptides of the invention are operatively linked to one or more regulatory sequences, which are capable of integrating the desired DNA molecule into a prokaryotic host cell.
  • Cells which have been stably transformed by the introduced DNA can be selected, for example, by introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • a selectable marker gene can either be linked directly to a nucleic acid sequence to be expressed, or be introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins described herein. It would be apparent to one of ordinary skill in the art which additional elements to use.
  • Factors of importance in selecting a particular plasmid or viral vector include, but are not limited to, the ease with which recipient cells that contain the vector are recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • the vector(s) may be introduced into an appropriate host cell by one or more of a variety of suitable methods that are known in the art, including but not limited to, for example, transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate-precipitation, direct microinjection, etc.
  • recombinant proteins can be detected by immunoassays including Western blot analysis, immunoblot, and immunofluorescence. Purification of recombinant proteins can be carried out by any of the methods known in the art or described herein, for example, any conventional procedures involving extraction, precipitation, chromatography and electrophoresis. A further purification procedure that may be used for purifying proteins is affinity chromatography using monoclonal antibodies which bind a target protein. Generally, crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized. The protein usually binds to the column via the specific antibody while the impurities pass through. After washing the column, the protein is eluted from the gel by changing pH or ionic strength, for example.
  • the efficacy of the treatment is evaluated by measuring, for example, the biological function of the treated organ (e.g., neuronal function).
  • the biological function of the treated organ e.g., neuronal function
  • a method of the present invention increases the biological function of a tissue or organ by at least 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or even by as much as 300%, 400%, or 500%.
  • the tissue is neuronal tissue and, preferably, the organ is brain.
  • the therapeutic efficacy of the methods of the invention is assayed by measuring an increase in cell number in the treated tissue or organ as compared to a corresponding control tissue or organ (e.g., a tissue or organ that did not receive treatment).
  • a corresponding control tissue or organ e.g., a tissue or organ that did not receive treatment.
  • cell number in a tissue or organ is increased by at least 5%, 10%, 20%, 40%, 60%, 80%), 100%), 150%), or 200% relative to a corresponding tissue or organ.
  • Methods for assaying cell proliferation are known to the skilled artisan and are described, for example, in Bonifacino et al., (Current Protocols in Cell Biology Loose-leaf, John Wiley and Sons, Inc., San Francisco, Calif).
  • assays for cell proliferation may involve the measurement of DNA synthesis during cell replication.
  • DNA synthesis is detected using labeled DNA precursors, such as [ 3H ]-Thymidine or 5-bromo-2*-deoxyuridine [BrdU], which are added to cells (or animals) and then the incorporation of these precursors into genomic DNA during the S phase of the cell cycle (replication) is detected (Ruefli-Brasse et al., Science 302(5650): 1581- 4, 2003; Gu et al., Science 302 (5644):445-9, 2003).
  • labeled DNA precursors such as [ 3H ]-Thymidine or 5-bromo-2*-deoxyuridine [BrdU]
  • kits for the treatment or prevention of a lysosomal storage disease or disorder e.g., Neuronal Ceroid Lipofuscinoses
  • a lysosomal storage disease or disorder e.g., Neuronal Ceroid Lipofuscinoses
  • the kit includes a composition containing an isolated hematopoietic stem cell expressing one or more
  • the kit includes a nanoparticle for ablative conditioning of endogenous microglial cells.
  • the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of the disease or disorder.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a neurological disease or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 Disease model selection for preclinical testing of Metallothioneins as therapeutic agents in LSDs
  • MT immunoreactivity was documented in all the tested LSD brains (FIG. 1A). MT signal was mostly detected in the grey matter of all the LSD brain samples, including the MLD and GLD ones. Astrocytes acted as primary MT-over-expressing cells in every tested sample. In the MLD samples MT signal was also identified in histiocytic cells. Interestingly, MT
  • INCL disease platform of potential value to test MT neuroprotective role in LSDs, and in particular we employed the INCL animal model.
  • GLD due to Galactocerebrosidase - GALC deficiency
  • MTs were shown to vary along with disease progression and upon therapeutic treatment (Cesani M. et al., Ann Neurol 2014; 75(1)).
  • Example 2 Phenotypic effects of Metallothioneins in the GLD and INCL animal models
  • MTtg MT-1 over-expressing transgenic mouse bearing multiple copies of the MT-1 gene (Palmiter RD. et al., Mol Cell Biol 1993; 13(9)) and expressing high MT-1 transcript levels both in the brain and peripheral tissues was taken advantage of (Comes G. et al., Int J Mol Sci 2017; 18(2); and data not shown).
  • MTtg mice were cross-bred with GLD and INCL heterozygous animals to generate GLD and INCL homozygous defective mice with constitutive high MT-1 levels in their tissues (MT-GLD and MT-INCL, respectively).
  • FIG. 2A High levels of MT-1 expression (FIG. 2A) were measured in the brain of these mice. Interestingly, in both settings the MT-1 expression levels resulted from the contribution of both the MT transgenic background and the disease setting per se. MT signal in the disease transgenic brains mostly co-localized with astrocytes, in line with the preferentially described expression pattern in the brain (Vela JM et al., Brain Res 1997; 767(2)) (FIG. 2B), and only a fraction of the microglia cells showed MT immunostaining (FIG. 2C). Interestingly, MT over- expression determined a therapeutic benefit in both the examined models.
  • MT-GLD transgenic mice showed a significantly increased survival as compared to not-transgenic affected GLD controls (approximately 10%, with a median survival of 40.5 days for MT-GLD and 37.5 days for GLD), which have a very short life expectancy and an extremely severe phenotype (FIG. 2D).
  • MT-INCL mice showed an increased survival compared to not-transgenic, affected INCL mice (FIG. 2E) (approximately 5%, with a median survival of 248 days in MT- INCL and of to 235.5 days in INCL), as well as an amelioration of their phenotype (FIG. 2F), as documented by a disease specific severity score.
  • MT expression levels were plotted against survival data collected in both LSD and MT-LSD mice (FIGS. 2G and 2H). Analysis of these data showed that MT levels equal or superior to 10- 20 folds the wild type MT expression levels are associated with increased survival of MT-INCL and MT-GLD mice versus the corresponding INCL and GLD animals, respectively (FIGS. 2G and 2H). Since both disease models in their original background are characterized by disease- associated high MT expression in the brain, this threshold for benefit translates into the need to increase the MT expression level of 1.5 to 2 folds on top of basal disease levels for GLD and INCL, respectively (FIGS. 2G and 2H).
  • Example 3 Modulation of anti-inflammatory, anti-apoptotic and anti-oxidative stress genes in MT-GLD and MT-INCL mice
  • genes were over-expressed in MTtg samples as compared to WT and they were mostly non- coding transcripts included in the Affymetrix platform.
  • the genes down-regulated in the MT- GLD model versus GLD identified functions related to neurotransmitter receptor activity, ion channel activity (voltage-gated potassium and calcium channels), transport channels, and others, indicating a possible new mechanism of MT action regulating brain network stability.
  • Other genes involved in inflammatory and apoptotic pathways were significantly down-regulated in MT-GLD versus GLD mice in the transcriptome array (FIG. 3B, FIG. 3C, FIG. 3D). These genes were validated through qPCR (FIG. 3B, FIG. 3C, FIG. 3D).
  • Ifi44 interferon- induced protein 44
  • UPGD - hydroxyprostaglandin dehydrogenases are potent mediators of several biological processes, including inflammation and oxidative stress (Nakao R.
  • nitrotyrosine was measured, a marker of cell damage, inflammation, and nitric oxide production, in the brain of MT-GLD and control mice through immunofluorescence (FIG. 3E and 3F) (Hidalgo J. et al., Exp Biol Med (Maywood) 2006; 231(9)). Nitrotyrosine signal was
  • MT-GLD brain cells also showed a tendency towards ROS reduction when stained for DCFDA (DCFDA 6- carboxy-2',7'-dichlorodihydrofluorescein diacetate) (FIG. 3G).
  • DCFDA DCFDA 6- carboxy-2',7'-dichlorodihydrofluorescein diacetate
  • Purkinje cell loss is known to contribute to the severe and complex phenotype of GLD, phenomenon that is known to be strictly linked to apoptosis (Lin DS et alGene 2015; 571(1)). It was confirmed that Purkinje cells are progressively lost from an early symptomatic to severely symptomatic stage in GLD mice. Importantly, prevention of Purkinje cell loss was observed in the brain of MT- GLD mice (FIG. 4A and FIG. 4B). This rescue was demonstrated through both qualitative and quantitative measures of Calbindin Parvalbumin signals on GLD and MT-GLD cerebellum slices obtained at advanced disease stage (FIG. 4C and FIG. 4D).
  • Purkinje cell bodies and dendritic arborizations representing early neuronal loss were also observed in the brain of MT-INCL mice already at intermediate disease stage (200 days), suggesting a specific effect of MTs on this neuronal compartment (FIG. 4E and FIG. 4F).
  • Example 5 Metallothionein over-expression is not impacting primary disease defects
  • MT over-expression was here shown to modulate secondary disease aspects of the two LSD models analyzed.
  • the primary disease mechanism is represented by accumulation of storage material due to the disease-causing lysosomal hydrolase defect.
  • MTs were not significantly affecting intracellular galactolipid storage in all brain regions analyzed in MT-GLD animals (FIG. 4G).
  • MT-INCL model we did not observe any changes in the accumulation of autofluorescent storage material in the experimental groups analyzed by flow cytometry and immunofluorescence analysis (FIG. 4H).
  • Example 6 Metallothioneins induce an anti-inflammatory M2 -like microglia phenotype in GLD and INCL
  • MTs could affect neuroinflammation in GLD and INCL mice.
  • Immunofluorescence for astrocyte and microglia markers revealed similar levels of astrogliosis and microgliosis in MT-LSD mice and naive LSD animals analysed at intermediate (MT-INCL and INCL) and advanced (PND36, MT-GLD and GLD) disease stage (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D), indicating that MTs, despite being mostly produced by the astrocytes, may not be able to affect the phenotype of these cells.
  • myeloid cells/microglia populations were isolated from MT-GLD and control mouse brains and quantified mRNA levels of some well-known markers of microglia activation in these samples.
  • Arginasel, CD206 and YM1 also referred as M2 myeloid cell markers was detected in the MT- GLD and MT-INCL cells over control GLD and INCL samples (FIG 5E, FIG 5F, FIG. 5G).
  • Example 7 Metallothioneins delivery by AAV vectors ameliorates the GLD phenotype
  • the minimum fold increase associated to a gain in survival was of 3.3 folds over basal GLD-associated MT transcript levels, in a range consistent with what observed in the MT-LSD models.
  • survival gain was associated to the down-regulation of inflammation, oxidative stress, and apoptosis genes in the AAV treated mice, as observed in MT-GLD mice (FIG. 6E, FIG. 6F, FIG. 6G), Without being bound by theory, this indicates that MTs were affecting the same pathway regulation mediated also upon exogenous AAV-mediated delivery.
  • MTs were previously identified and characterized as biomarkers of brain disease that dynamically modify their expression in the course of disease progression and response to treatment in a variety of LSDs (Cesani M. et al., Ann Neurol 2014; 75(1): 127-137). MTs could exert a protective role in the diseased brain, since their expression levels in astrocytes increase upon administration of anti-inflammatory drugs. MTs were shown to have neuroprotective capacities in acute brain damage (West AK et al., Neurotoxicology 2008; 29(3)) and more recently in chronic diseases, as Parkinson's disease (Ebadi MH et al., Brain Res Mol Brain Res 2005; 134(1)), Amyotrophic Lateral Sclerosis (Tokuda E.
  • MTs are described to exert anti-oxidant, anti-inflammatory and anti-apoptotic functions in the diseased brain, but little is known about MT-activated pathways and their mechanisms of action in this compartment (Ito Y. et al., Curr Pharm Biotechnol 2013; 14(4)).
  • Experimental evidences support the hypothesis that MTs, synthesized from astrocytes in the diseased brain upon pro-inflammatory and pro-oxidative stimuli, can be up-taken by neurons through the receptor Lrp2/Megalin and then exert their detoxifying activity (Chung RS et al., J Biol Chem 2008; 283(22)), mostly in the context of neuronal damage.
  • MTs One of the most striking effects of MTs is the reduction of oxidative stressand related pathways, as it was shown in a mouse model of dystrophinopathy(Di Foggia V. et al., J Exp Med 2014; 211(13)). It is also widely accepted that a dysregulation of different pathways involved in oxidative stress responses and inflammation occurs in LSDs as consequence of a block of autophagy (Settieri C. et al., Nat Rev Mol Cell Biol 2013; 14(5)). Finally, MTs were shown to protect against oxidative stress-induced lysosomal destabilization (Baird SK. et al., Biochem J 2006; 394(Pt 1)).
  • MT could be over- expressed in response to oxidative stress and inflammation caused by lysosomal dysfunction.
  • the therapeutic effects exerted by MTs in the LSD models could be mediated by their ability to modulate these events.
  • measurements performed in our models indicate that MTs can down-modulate and mitigate oxidative stress associated to lysosomal dysfunction, being potentially beneficial to the damaged Purkinje layer.
  • the possible impact of MTs on the M1/M2 balance in the MTtg-LSD brains is also further confirmed by the evidence of a reduction of oxidative stress in microglia cells from MT-GLD mice, which are endowed with M2 features, phenomenon that is widely described in literature (Rojo AL et al., Antioxid Redox Signal 2014; 21(12)).
  • the involvement of microglia in mediating MT-driven neuroprotection is reported here for the first time.
  • AAV- PHP.B vectors were generated containing one or four MT copies linked by three different 2A peptides, and confirmed the greater performance of the expression system with four copies in driving increased MT expression both at the RNA and protein levels.
  • this AAV-4MT vector reproduced and validated the findings observed in MTtg-GLD mice.
  • AAV-mediated delivery of MT-1 cDNA and its expression in the affected GLD brain exerted similar effects as constitutive expression by transgenesis, with significantly improved survival, modulation of inflammation and oxidative stress, and anti-apoptotic effects exerted in the GLD central nervous system.
  • MT supplementation therapy is envisaged in any form available in terms of clinical translation as a neuroprotective strategy that may be eventually coupled with other approaches aiming at enzyme activity reconstitution.
  • mice harboring murine MT-1 (strain B6.Cg-Tg(Mtl)174Bri/J, stock number 002210) were purchased from the Jackson Laboratory. MT-1 mice were maintained on a
  • mice were isolated and fixed for 16 hours in 4% paraformaldehyde, equilibrated in 10-30% sucrose gradient in PBS for 48 hours and then embedded in OCT compound for quick freezing. 16-micron cryostat sections were incubated overnight at 4°C with primary antibodies: mouse monoclonal to Metallothionein (DAKO) 1 : 100; rabbit anti-glial fibrillary acidic protein (GFAP) (MCA1909; Serotec Ltd) 1 :500; rabbit anti-Ibal (Wako) 1 : 100, mouse monoclonal anti-Nitrotyrosine (Abeam) 1 :500; rat anti- mouse CD206 (AbD Serotec) 1 :200; rabbit anti-Calbindin (Swant) 1 :700; rabbit anti- Parvalbumin (Swant) 1 :700; then for 1 hour 30 minutes at RT with secondary antibodies: goat anti-mouse Alexa Fluor488 1 : 1000; rabbit anti-goat Alexa Fluor488 1
  • ROS reactive oxygen species
  • RNA extraction RNA from sorted microglia was extracted with RNeasy plus Micro Kits (Qiagen), RNA from cerebella (20C ⁇ g) used for Whole Transcriptome Assay was extracted with RNeasy Lipid Tissue Mini Kit and treated with DNase I (Qiagen). Quantitative PCR was performed for the following genes: MT1 Mm00496660_gl, ILlp Mm00434228_ml, TNFa Mm00443258_ml, Ifi44 Mm00505670_ml, Arginase Mm00477592_ml, CD206
  • RNA microarray hybridization and image acquisition, processing and bioinformatics analysis were performed .
  • Hybridization was performed using the GeneChip® Hybridization, Wash and Stain Kit that contains mix for target dilution, DMSO at a final concentration of 7% and pre-mixed biotin-labelled control oligo B2 and bioB, bioC, bioD and ere controls (Affymetrix cat #900299) at a final concentration of 50 pM, 1.5 pM, 5 pM, 25 pM and 100 pM, respectively.
  • Targets were diluted in hybridization buffer at a concentration of 25 ng/ ⁇ , denatured at 99 °C for 5 minutes, then incubated at 45 °C for 5 minutes and centrifuged.
  • Transcriptome Array 1.0 was then hybridized with each biotin-labelled sense target.
  • Hybridizations were performed for 16 h at 45 °C in a rotisserie oven. GeneChip® cartridges were washed and stained with GeneChip®. Hybridization, Wash and Stain Kit in the Affymetrix Fluidics Station 450 following the FS450 0002 standard protocol, including the following steps: (1) (wash) 10 cycles of 2 mixes/cycle with Wash Buffer A at 30 °C; (2) (wash) 6 cycles of 15 mixes/cycle with Wash Buffer B at 50 °C; (3) stain of the probe array for 5 min in SAPE solution at 35 °C; (4) (wash) 10 cycles of 4 mixes/cycle with Wash Buffer A at 30 °C; (5) stain of the probe array for 5 min in antibody solution at 35 °C; (6) stain of the probe array for 5 min in SAPE solution at 35 °C; (7) (final wash) 15 cycles of 4 mixes/cycle with Wash Buffer A at 35 °C; (8) fill the probe array with Array Holding buffer.
  • GeneChip arrays were scanned using an Affymetrix GeneChip® Scanner 3000 7G using default parameters.
  • Affymetrix GeneChip® Command Console software was used to acquire GeneChip® images and generate .DAT and .CEL files, which were used for subsequent analysis with proprietary software.
  • a cassette encoding either one or four copies of MT-1 separated by 2 A peptides was cloned in place of GFP in the AAV-CBA.GFP-Wpre plasmid.
  • AAV-PHP.B vectors were produced by transient triple transfection of HEK- 293 cells with transfer plasmid, Fd6 helper plasmid and a plasmid generated in the Sena-Esteves laboratory carrying AAV2 rep and the recently described AAV-PHP.B cap genel3.
  • Vectors were purified by iodixanol gradient centrifugation followed by buffer exchange to phosphate buffered saline (PBS) using 7K MWCO Zeba Spin Desalting columns (Thermo Scientific) and finally concentrated with 100K Amicon Ultra- 15 centrifugal filters (Merck Millipore, Cork, Ireland). Titers were determined by qPCR using primers and probes to the BGH polyadenylation signal.
  • AAV vectors were injected via the superficial temporal vein of two-day old (P D2) GLD mice as described (Capotondo et al, 2017, submitted). Control mice received PBS.

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Abstract

La présente invention concerne des compositions et des procédés pour le traitement ou la prévention d'une maladie ou d'un trouble lysosomial impliquant l'augmentation du niveau, de l'expression ou de l'activité d'un polypeptide ou d'un polynucléotide de métallothionéine chez le sujet.
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WO2021224633A1 (fr) 2020-05-06 2021-11-11 Orchard Therapeutics (Europe) Limited Traitement pour les maladies neurodégénératives

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